Congenital malformations are a major source of human morbidity and mortality. The role of chemical exposure and other environmental factors in the etiology of congenital malformations is not completely understood. However, many xenobiotic chemicals are known to be developmental toxicants or teratogens in experimental animals and several human birth defects are associated with embryonic exposure to xenobiotics. Despite this understanding, the mechanisms by which most developmental toxicants and teratogens disrupt embryonic development are not known. In this R21 (Exploratory/Developmental Research) grant application, we propose to investigate a new potential mechanism of developmental toxicity-disruption of microRNA expression-and to establish the zebrafish embryo as a model for studying the roles of microRNAs in developmental toxicology and teratogenicity. MicroRNAs are single-stranded RNA molecules of ~22 nucleotides that regulate the expression of mRNAs by inhibiting their translation into proteins and promoting their sequestration or degradation. MicroRNAs have essential roles during embryonic development;disruption of microRNA expression or function in embryos can cause developmental abnormalities involving a variety of tissues. In adult animals, microRNAs also are involved in regulating cellular responses to xenobiotic chemicals. Together, these results suggest that altered expression of microRNAs might underlie some effects of developmental toxicants and teratogens. However, the possible role of microRNAs in developmental toxicology and teratology has not yet been explored. Zebrafish embryos have been instrumental in some of the fundamental advances in understanding the roles of microRNAs in embryonic development in vertebrates. Thus, the zebrafish embryo is an excellent model with which to test whether altered expression of microRNAs is an important mechanism of developmental toxicity. In aim 1, we will test the hypothesis that the developmental toxicity of the well-known developmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish embryos is accompanied by altered expression of microRNAs. We will compare two methods for microRNA expression profiling: microRNA microarrays using locked nucleic acid (LNA) probes, and deep sequencing (pyrosequencing / 454 technology). In aim 2, we will test the hypothesis that distinct patterns of altered microRNA expression occur after exposure to developmental toxicants that act via different mechanisms. We will choose one microRNA profiling method based on results of Aim 1 and perform microRNA profiling in zebrafish embryos exposed to the known human teratogens ethanol, valproic acid, and retinoic acid. These exploratory experiments will take advantage of new information on microRNA-dependent mechanisms of development and new deep sequencing technologies to provide data that could transform our understanding of mechanisms involved in developmental toxicology. PUBLIC HEALTH RELEVANCE: Congenital malformations (birth defects) are a major source of human morbidity and mortality. The role of chemical exposure and other environmental factors in contributing to congenital malformations is not completely understood. However, it is known that many drugs and environmental chemicals are developmental toxicants or teratogens in experimental animals and that several human birth defects are strongly associated with embryonic exposure to these compounds. Despite this understanding, the mechanisms by which most developmental toxicants and teratogens disrupt embryonic development have not yet been fully elucidated. Here we propose to investigate new potential mechanism of developmental toxicity- disruption of microRNA expression. MicroRNAs are single-stranded RNA molecules of ~22 nucleotides that regulate the expression of messenger RNAs by inhibiting their translation into proteins and promoting their degradation. Recent research has established that many microRNAs have critical roles during embryonic development. We hypothesize that some developmental toxicants and teratogens may act by disrupting microRNA expression. The proposed research will establish the zebrafish embryo as a valuable model for studying the roles of microRNAs in developmental toxicology.